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Abstract

Introduction

Disease severity in collagen-induced arthritis (CIA) is commonly assessed by clinical
scoring of paw swelling and histological examination of joints. Although this is an
accurate approach, it is also labour-intensive and the application of less invasive
and less time-consuming methods is of great interest. However, it is still unclear
which of these methods represents the most discriminating measure of disease activity.

Methods

We undertook a comparative analysis in which different measurements of inflammation
and tissue damage in CIA were studied on an individual mouse level. We compared the
current gold standard methods - clinical scoring and histological examination - with
alternative methods based on scoring of X-ray or micro-computed tomography (CT) images
and investigated the significance of systemically expressed proteins, involved in
CIA pathogenesis, that have potential as biomarkers.

Results

Linear regression analysis revealed a marked association of serum matrix metalloproteinase
(MMP)-3 levels with all features of CIA including inflammation, cartilage destruction
and bone erosions. This association was improved by combined detection of MMP-3 and
anti-collagen IgG2a antibody concentrations. In addition, combined analysis of both
X-ray and micro-CT images was found to be predictive for cartilage and bone damage.
Most remarkably, validation analysis using an independent data set proved that variations
in disease severity, induced by different therapies, could be accurately represented
by predicted values based on the proposed parameters.

Conclusions

Our analyses revealed that clinical scoring, combined with serum MMP-3, anti-collagen
IgG2a measurement and scoring of X-ray and micro-CT images, yields a comprehensive
insight into the different aspects of disease activity in CIA.

Introduction

The systemic autoimmune disease rheumatoid arthritis (RA) is characterized by synovial
inflammation followed by progressive destruction of articular cartilage and subchondral
bone. Mouse models are often used to gain further insight into the pathological mechanisms
of joint inflammation as well as for preclinical evaluation of therapeutic agents.
In this context, collagen induced arthritis (CIA) is the most widely studied animal
model for RA as it models the similarities in pathology and immunological processes
involved in the disease [1].

Currently, clinical scoring of inflamed paws over time is the standard method used
for quantification of disease severity. This is followed by histological examination
of joints to assess inflammation, and cartilage and bone loss. More recently, alternative
and less time-consuming techniques such as scoring of X-ray and micro-computed tomography
(micro-CT) images have been employed. The Larsen score is well established for the
scoring of X-ray pictures and accounts for abnormalities of the joint space and bone
erosions [2,3]. Alternatively, when performing micro-CT analysis, the number of objects per micro-CT slice parameter can be used as an indicator of bone porosity [4].

Moreover, the application of biomarkers that are measurable in serum, urine or tissue
has become a topic of increasing interest. Pro-inflammatory cytokines like tumour
necrosis factor-α (TNF-α) are detected in arthritic mice and cartilage degradation
products such as cartilage oligomeric matrix protein (COMP) and C-telopeptide fragments
of type II collagen (CTXII) have been described as potential biomarkers for cartilage
destruction in CIA [5-7]. Furthermore, the production of autoantibodies to type II collagen (CII) is a typical
feature of CIA [8]. Despite the fact that these methods are regularly used, it remains unclear which
read-out parameters are most useful when assessing CIA.

We therefore established an analysis platform in which the degree of inflammation
and tissue damage in CIA was assessed at the level of individual mice. This platform
incorporated different scoring methods including histological examination and techniques
based on scoring of X-ray or micro-CT images. Furthermore, systemically expressed
proteins involved in CIA pathogenesis were investigated for their potential as biomarkers.

We report here the findings of our search for useful scoring methods and valuable
biomarkers to monitor different aspects of disease severity in CIA. First, we can
conclude that a combined analysis of scoring X-ray's using a modified Larsen score
and quantification of erosions on the calcaneus as visualized by micro-CT imaging
is predictive for both cartilage damage and bone erosions in CIA. Out of the selected
proteins in our study, a combined analysis of serum MMP-3 and anti-collagen IgG2a
antibody concentrations turned out to be indicative not only for the inflammatory
aspect of CIA but also for cartilage and bone destruction, emphasizing the inflammatory
nature of the disease. Most important, in an independent experiment, this set of proposed
parameters is capable of accurately representing variations in disease severity induced
by different therapies. Taken together, this critical analysis of read-outs for monitoring
disease activity and therapeutic responses in the CIA model has led to the identification
of parameters that allow faster analysis of treatment efficacy with minimal loss of
information.

Materials and methods

Mice

Male, 9 to 10-week-old DBA/1 mice were purchased from Janvier (Le Genest Saint Isle,
France) and housed following institutional guidelines. Experiments were conducted
according to the guidelines of the Ethics Committee of Laboratory Animals Welfare
of Ghent University.

Induction and analysis of CIA

Mice were immunized intradermally at the base of the tail with 200 μg of chicken type
II collagen (CII) (Morwell Diagnostics GmbH, Zurich, Switzerland) (in 0.1 M acetic
acid) emulsified in Incomplete Freund's Adjuvant + mycobacterium Tuberculosis H37RA
(150 μg/mouse) (Difco, Lawrence, KS, USA). Twenty-one days later, mice were re-challenged
with an injection of CII in Incomplete Freund's Adjuvant. From Day 21, mice were monitored
for clinical symptoms of arthritis until the day of sacrifice (Day 42). Clinical severity
was graded as follows: 0 = normal; 0.5 = erythema and edema in only one digit; 1 =
erythema and mild edema of the footpad, or ankle or two to five digits; 2 = erythema
and moderate edema of two joints (footpad, ankle, two to five digits); 3 = erythema
and severe edema of the entire paw; 4 = reduced swelling and deformation leading to
incapacitated limb.

The individual mouse arthritic score was obtained by summing the scores recorded for
each limb. Clinical evaluations were performed by two investigators unaware of mouse
identity and the mean of both scores was calculated.

Scoring of X-ray images

Prior to histology, anteroposterior X-ray radiographs were taken (Faxitron M20, Edimex,
France) of one hind paw per mouse. The severity of bone erosion was blindly ranked
by three independent scorers using a modified version of the Larsen scoring method:
0 = normal; 1 = slight abnormality with any one or two of the exterior metatarsal
bones showing slight bone erosion; 2 = definite early abnormality with any of the
metatarsal or tarsal bones showing bone erosion; 3 = medium destructive abnormality
with the metatarsal bones or any one or two of the tarsal bones showing definite bone
erosions; 4 = severe destructive abnormality with all the metatarsal bones showing
definite bone erosion and at least one of the tarsometatarsal joints being completely
eroded, leaving some bony joint outlines partly preserved; 5 = mutilating abnormality
with no bony outlines that can be deciphered.

Histological evaluation

To allow comparison between the different techniques, histology was performed on the
same paws that were used for X-ray and micro-CT imaging. Knees and paws were fixed
in 4% formaldehyde, decalcified and embedded in paraffin. Serial sections of the knee
were stained with hematoxylin and eosin (H&E) or with saffranin O-fast green and inflammation
and joint damage of the femorotibial joint were investigated by scoring five parameters
as follows: inflammation was scored on a scale of 0 (no inflammation) to 3 (severe
inflamed joint) depending on the number of inflammatory cells in the synovial cavity
(exudate) and synovial tissue (infiltrate). Exudate and inflammatory infiltrate were
both assigned individual scores. Loss of proteoglycans was scored on a scale of 0
to 3, ranging from fully stained cartilage to destained cartilage or complete loss
of articular cartilage. Cartilage destruction was scored on a scale of 0 to 3, ranging
from the appearance of dead chondrocytes (empty lacunae) to complete loss of the articular
cartilage. Loss of bone was scored on a scale of 0 to 5 ranging from no damage to
complete loss of the bone structure [9]. We also stained serial sections of the paw and examined the following parameters:
pannus severity, infiltrate, cartilage lesion and bone lesion. The scoring system
ranged from 1 to 4: 1 = normal, 2 = mild, 3 = moderate, 4 = severe.

For both knee and paw histology, a composite score was calculated by summing the individual
parameters. Scoring was executed blindly by two investigators and mean values were
calculated.

Statistical analysis

For analysis of longitudinal clinical scores, mixed model analysis with random intercept
was used. Differences in clinical (day of onset and Day 42 score) and histological
data between the treatment groups were assessed by Kruskal Wallis tests followed by
Mann-Whitney-U test with correction using the Holm procedure. Fisher's Exact test
was applied to analyse arthritis frequencies. Stepwise linear regression analysis
was performed with the scoring methods, serum markers and treatment group as covariates.
All analyses were performed using SPSS 15.0 statistical software (Chicago, IL, USA).

Results

Evaluation of CIA by clinical and histological examination

We initiated this study by examining CIA severity using the current gold standard
method: clinical scoring followed by histological analysis. To create gradations in
disease severity, mice were treated semi-therapeutically with one of four different
therapies: dexamethasone, etanercept, zoledronic acid or abatacept. Vehicle (PBS)
administration was used as a negative control. Pooled data of two independent experiments
are shown in Figure 1A and Table 1. As expected, semi-therapeutic treatment of mice with dexamethasone completely abolished
the induction of CIA. Also etanercept induced significant improvement in arthritis
symptoms. Treatment with abatacept on the other hand resulted in a small, albeit not
significant, reduction in disease severity and one-time administration of zoledronic
acid showed no protective effect.

Figure 1.A) Clinical scoring of arthritis symptoms in CIA. From Day 20 after CIA induction, mice received semi-therapeutic treatment with one
of four therapies: dexamethasone (n = 23), etanercept (n = 23), zoledronic acid (n = 24) or abatacept (n = 23). Control mice were treated with the vehicle PBS (n = 24). Signs of arthritis were monitored until Day 42. Significant reduced arthritis
scores were observed for dexamethasone and etanercept treatment as compared to PBS
administration (mixed model analysis, random intercept, P < 0.001 for both dexamethasone and etanercept). Means + SEM of pooled data from two
independent experiments are depicted. B) Histological examination of arthritis symptoms in the knee joint of CIA mice. At Day 42 post-immunization, knee joints were isolated and prepared for histological
examination. Femorotibial joints were scored for five different parameters. Kruskall-Wallis
test: P ≤ 0,001 for all parameters as compared to PBS. *: significant P-values (compared to PBS) according to Mann-Whitney U test and correction using the
Holm procedure. Bars represent means + SEM.

In contrast to clinical scoring, which is mainly indicative of the degree of inflammation,
histological examination provides additional information pertaining to cartilage and
bone damage. In line with the absence of clinical symptoms, histological scoring of
the knee joint revealed no signs of inflammation (inflammatory infiltrate and exudate),
proteoglycan depletion, destruction of articular cartilage or bone erosion in dexamethasone
treated animals (Figure 1B). Although the composite score of knee histology was borderline not significant after
statistical correction using the Holm procedure (P = 0.019 > 0.017 (Holm)), the attenuated clinical disease activity observed in mice
treated with etanercept was in agreement with strong reductions in the five parameters
analysed when compared to vehicle treated mice. Comparable to a rather small improvement
of clinical arthritic symptoms in the abatacept treated mice, all histological parameters
investigated showed a moderate reduction in severity. Treatment with zoledronic acid
did not induce improvement in any of the histological factors. In the context of the
four therapies used, similar trends were observed between the histological assessment
of disease and the clinical score.

Beside the knee joint, also hind paw joints are commonly subjected to histological
examination and in the present study, we examined the correlation between both techniques
and the clinical score. Based on our results, it can be concluded that although clinical
scoring addresses paw inflammation, histological examination of the knee joint correlates
slightly better with the clinical scoring as compared to paw histological scoring
(r = 0.725 and r = 0.533 for histology composite scores of respectively knee and paw
versus clinical scoring on Day 42). These findings highlight the fact that CIA is
a systemic disease and that both sites are useful for histological examination.

Scoring of plain X-ray and micro-CT images are valuable methods to assess cartilage
and bone damage in CIA

Despite the fact that histology provides additional information on cartilage and bone
damage, the procedure is time-consuming and only allows cross-sectional analysis.
We therefore compared the use of two non-invasive scoring techniques based on X-ray
and micro-CT imaging of paws, with both clinical and histological scoring. For scoring
of plain X-ray images, a modified Larsen score was applied on hind paws of the mice.
On reconstructed three-dimensional micro-CT images, the focal bone erosion at the
calcaneus is clearly visible (Figure 2). This bone erosion was quantified by counting the average number of objects per slice on the 2 D micro-CT images of the calcaneus, using image analysis software.

For further analysis, stepwise linear regression modelling was performed on data from
a first experiment. Linear regression analysis, using a clinical score on Day 42 as
the dependent variable and composite scores for knee and paw histology, Larsen score,
micro-CT calcaneus score and treatment group as independent variables, indicates that
from these four different scoring methods, clinical scoring is most accurately represented
by the composite score of knee histology (adjusted R2 = 0.464). Despite this strong correlation between the clinical score and knee histology,
we opted to investigate the capacity of X-ray or micro-CT imaging, performed on paws,
to predict joint damage, in relation to histology of the paw. Therefore, two linear
regression analyses with either the cartilage or bone parameter of the paw histology
as dependent variable were fitted (Table 2). Results suggested that the Larsen score was most indicative for cartilage destruction
in the paw, but the predictive value of the regression model was greatly enhanced
by addition of the calcaneus erosion score and the clinical score (R2 = 0.704). However, for the detection of bone erosions, the calcaneus score showed
the best correlation with histology yet, combined analysis of calcaneus and Larsen
score was much more predictive for bone erosions (R2 = 0.713). These results indicate that a combined analysis of clinical score, X-ray
Larsen score and micro-CT calcaneus score provide useful information regarding cartilage
and bone destruction in CIA.

MMP-3 is related to inflammation as well as cartilage and bone damage in CIA

For the identification of biomarkers to assess disease severity in CIA, the concentrations
of 10 serum proteins were assessed at the time of sacrifice. The selected proteins
constitute inflammatory mediators and chemokines involved in arthritis, including
TNF-α, IL-17, IL-6, MMP-3 and KC. Additionally, indicators of cartilage degradation
(COMP and CTXII) and bone damage (CTXI) were determined as well as the presence of
antibodies to type II collagen (IgG1 and IgG2a). As serum levels of IL-6 were mostly
undetectable, this cytokine was eliminated from further analysis.

Although analysis on the individual biomarker level revealed significant correlations
with clinical score, cartilage destruction and bone erosion for five out of nine proteins
(COMP, CTXII, CTXI, anti-collagen IgG2a and MMP-3) (data not shown), only three of
them contributed significantly in the performed linear regression models. No significant
correlations were observed for the investigated cytokines (IL-17 and TNF-α). In three
models with clinical score, or cartilage or bone destruction of the knee histology
as dependent variables, we incorporated all nine proteins, including the cytokines,
and the treatment group (Table 3). Remarkably, measurement of MMP-3 concentrations in serum was found to be most indicative
for clinical score. Analysis of histological parameters revealed that MMP-3 is not
only a good indicator for the inflammatory aspect of CIA but also for cartilage and
bone damage. Addition of anti-collagen IgG2a to the models improved the association
with clinical score and bone destruction. Combined detection of both MMP-3 and CTXII
on the other hand was more indicative for cartilage destruction.

Validation of the proposed regression models

To validate the obtained results, the data set from a second independent experiment
was fit in the regression equations derived from the initial analyses, resulting in
predicted values. Because linear regression analysis, combining scoring methods and serum markers,
did not yield important contributions for serum markers in predicting cartilage and
bone degradation (Table 4), these aspects of the disease were only validated using X-ray and micro-CT analysis.
Clinical score on the other hand, was predicted using MMP-3 and anti-collagen IgG2a
antibody concentrations. In the validation analyses, we studied the capacity of the
predicted values to distinguish between the different therapies. As CIA experiments
are always performed on a group of animals and not on the individual level, we opted
to perform the validation analysis on the level of the treatment groups. Figure 3 clearly demonstrates that the same trends between the different treatment groups
can be detected in the graphical representations of both the observed and predicted
values for the clinical score, cartilage degradation and bone destruction. This was
confirmed by very strong correlations between the observed and predicted mean values
for each treatment group (Spearman's rho = 0.94, R = 0.99 and R = 0.99 for predicted
versus observed means of respectively clinical score, cartilage destruction and bone
erosions). These validation analyses provides compelling evidence that the proposed
read-out parameters, MMP-3, anti-collagen IgG2a, and Larsen and micro-CT calcaneus
score, are most applicable to detect changes in different aspects of arthritis severity
upon treatment.

Figure 3.Validation of the proposed regression models. Graphical reproduction of the observed and predicted values for clinical score,
cartilage destruction and bone erosions based on a second independent data set. Predicted
values were calculated using the linear regression equations obtained from the first
experiment. Bars represent means + SEM and r = Spearman's rho representing the correlations
between the observed and predicted mean values for each treatment group.

Discussion

As in many other human or experimental diseases, the field of surrogate analysis markers
is also in RA and its experimental models of increasing importance. Therefore, it
is surprising that a comprehensive analysis, aimed at assessing various read-out parameters
for monitoring disease activity is still lacking for CIA, the most commonly used animal
model of RA. In this report, we use linear regression analysis to propose a restricted
set of key indicators that allows the assessment of CIA in a more objective and less
labour-intensive way.

When evaluating the value of serum markers, MMP-3 was found to be highly indicative
of all the features of CIA, including inflammation, cartilage destruction and bone
erosion. This is in line with previous reports that demonstrated elevated MMP-3 expression
levels in synovial fluids and sera of patients with rheumatoid arthritis as well as
expression in joints of CIA rats [10-12]. Although several studies have proposed MMP-3 as a useful indicator for inflammation
in RA, the correlation of MMP-3 with joint destruction or its predictive value is
still a matter of debate [10,13-18]. Our results, however, showed a strong correlation of this marker with joint damage.
A second marker that also proved to be related to inflammation and bone destruction
is the anti-CII IgG2a antibody. Antibodies to CII are detected in both human as well
as experimental arthritis early in disease development and the IgG2 subclass is capable
of initiating inflammatory processes by activating the complement cascade [8,19-21].

Rather unexpected was the finding that, in combination with MMP-3 and anti-collagen
IgG2a, detection of cartilage or bone degradation products such as COMP and CTXI in
serum could not improve the predictive value of the regression models for cartilage
or bone destruction as assessed by knee histology. However, CIA is an experimentally
induced arthritis that is strongly mediated by inflammation. This could clarify why
inflammatory mediators rather than specific indicators of cartilage and bone degradation
proved to be better biomarkers in this animal model. In addition, since the serum
levels for the different markers were assessed at the end of the study, different
kinetics of the COMP or CTXI markers might represent another explanation for their
lower predictive value observed in this study. This possibility of different kinetics
could also provide an explanation for the fact that the inflammatory cytokines TNF-α
and IL-17 did not correlate with clinical score and joint damage.

We also evaluated the potential of different imaging techniques to assess disease
activity in CIA, which yielded a second set of important findings. First, scoring
of X-ray pictures using a modified Larsen score was most indicative for destruction
of cartilage. It should be mentioned though that cartilage itself cannot be visualised
by X-rays. Therefore, cartilage destruction is quantified indirectly by incorporating
joint space narrowing as a parameter in the scoring system. Second, for the assessment
of bone erosions, we introduced a rather new method that is based on micro-CT imaging
and allows objective and high throughput analyses. So far, bone loss on micro-CT was
mainly studied using parameters describing bone volume and composition [22,23]. A major hallmark of RA and CIA, however, is the appearance of focal bone erosions
[24] and to quantify these erosions we calculated the number of objects on slices through the calcaneus as a measure for bone fragmentation. Our analysis
showed that this method correlated well with the bone erosion score of histology.
Importantly, in both cases, combined analysis of X-ray Larsen and micro-CT calcaneus
score greatly enhanced the predictive values of the regression models for cartilage
and bone destruction. Both X-ray and micro-CT imaging allow longitudinal follow-up
of disease activity in animals. However, our analyses only take into account the read
out-parameters on Day 42 and as such, they do not allow us to draw conclusions about
the utility and sensitivity of these methods at earlier time points. A follow-up study
comparing the techniques at different time-points of disease progression could answer
this question. Additionally, it would also yield useful information about the predictive
value of the techniques and it could indicate if a correct analysis for the different
aspects of the disease severity in CIA could be obtained at an earlier time-point
to shorten the experiment time.

Remarkably, when linear regression analyses, containing both serum markers and scoring
methods, were performed, results demonstrated that serum markers did not increase
the predictive value of the models for cartilage and bone damage. This implies that
serum markers are inferior to imaging methods for the evaluation of disease activity
and therapeutic efficacy. One can however not conclude that a role for the investigated
serum markers in CIA is minimal. As already mentioned, serum levels of proteins may
display certain kinetic patterns in pathologies and as such, timing of the evaluation
could be important. Furthermore, serum concentrations of proteins do not necessarily
reflect the local situation in the joint. Investigation of biomarker expression in
the joint would yield useful information but these methods are often more invasive
and as such less applicable.

Validation of the obtained results by utilizing an independent data set, demonstrated
that values, predicted by the proposed read-out parameters, were also capable to differentiate
between various therapies with a different mode of action. The same trends between
the different treatment modalities could be observed in the graphical representations
of both the predicted values by Larsen and calcaneus score and the values observed
by histological examination. Since the last method is very time-consuming and only
allows cross-sectional analysis, combined analysis of X-ray and micro-CT images might
provide a worthy alternative. One should, however, also take in mind that the analyses
performed in our study focus on the utility of different read-out parameters to assess
disease severity in CIA. As different animal models exist to investigate rheumatoid
arthritis, for example, adjuvant-induced arthritis, zymosan induced arthritis and
so on, a comparable study should be performed for all the other models. Similarly,
it would be interesting to investigate the value of imaging techniques and serum markers
in other joint disorders, such as osteoarthritis. In other words it remains to be
determined whether imaging techniques and serum markers also apply in other models
of experimental arthritis.

Conclusions

We can conclude that MMP-3 is a strong indicator for the inflammatory features of
CIA. The fact that this marker is also related to cartilage and bone destruction emphasizes
the inflammatory nature of the disease. Furthermore, combined analysis of X-ray Larsen
and micro-CT calcaneus erosion score proved to be predictive for both cartilage and
bone destruction. Overall, our analyses revealed that clinical scoring, combined with
serum MMP-3 and anti-collagen IgG2a measurement and scoring of X-ray and micro-CT
images yield a comprehensive insight into the different aspects of disease activity
in CIA.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SS, PJ, JVP, MD, LO, NV, RB, GV and DE participated in the study design. SS, PJ, JVP,
JC, TD, LP, PP and LO participated in the acquisition of data. ED delivered support
for statistical analysis. SS, PJ, JVP and DE participated in the analysis and interpretation
of the data. SS and DE prepared the manuscript. All authors read and approved the
final manuscript.

Acknowledgements

We are thankful to Steve De Vos, An Van de Water, Didier Merciris and Maarten van
Balen for their input in the cytokine determinations an histological evaluation.

Part of this study was funded by the Flemish government (IWT-Vlaanderen, Institute
for the Promotion of Innovation through Science and Technology in Flanders IWT060235).
SS is a predoctoral researcher supported by a personal scholarship from IWT-Vlaanderen.
PJ, JVP and MD are supported by the Fund for Scientific Research-Flanders (FWO-Vlaanderen).